1. Volume 25, Issue 7, Pages 1718-1728.e4 (November 2018)
Two B-Box Domain Proteins, BBX18 and BBX23, Interact with ELF3 and Regulate Thermomorphogenesis in Arabidopsis 
Lan Ding, Shuo Wang, Ze-Ting Song, Yupei Jiang, Jia-Jia Han, Sun-Jie Lu, Lin Li, Jian-Xiang Liu 
Cell Reports 
Volume 25, Issue 7, Pages e4 (November 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 1718-1728.e4DOI: (10.1016/j.celrep.2018.10.060)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
BBX18 and BBX23 Are Positive Regulators of Thermomorphogenesis in Arabidopsis
(A) Upregulation of BBX18 and BBX25 by increased ambient temperature. Seven-day-old Arabidopsis wild-type (WT) seedlings grown at 22°C were transferred to either 22°C or 29°C for 3–9 hr, after which the expression of group IV BBX genes was examined by qRT-PCR. Fold induction is the expression level of each gene at 29°C normalized to that at 22°C, both of which were normalized to that of PP2A. The bars depict the SE (n = 3). ∗∗p < 0.01; ∗p < 0.05; #, not significant at p = 0.05 in Student’s t test.
(B and C) Increase in BBX18 and BBX23 protein levels in response to elevated ambient temperature. BBX18-FLAG (B) and BBX23-FLAG (C) fusion proteins with the constitutive CaMV 35S promoter were expressed in Arabidopsis plants. Seven-day-old transgenic seedlings grown at 22°C were transferred to either 22°C or 29°C for 3–9 hr, after which the fusion proteins were checked by western blotting with anti-FLAG antibodies. Tubulin served as a protein-loading control. Signal intensity of each band was quantified and normalized to that of the first sample.
(D and E) Short hypocotyl phenotypes of BBX18 or BBX23 loss-of-function mutant at an elevated temerature.
(F and G) Long hypocotyl phenotypes of BBX18 overexpression plants at an elevated temperature. BBX18-FLAG and BBX23-FLAG fusion proteins with the constitutive CaMV 35S promoter were overexpressed in Arabidopsis WT plants.
Four-day-old seedlings of the wild-type (WT), BBX18/BBX23 single or double mutant, PIF4 mutant (pif4-101), and BBX18ox and BBX23ox transgenic seedlings grown at 22°C were transferred to either 22°C or 29°C for 4 days, after which representative plants were imaged and the hypocotyl length of each plant was subsequently measured. Three biological replicates were performed, and eight plants were measured in each replicate. The bars depict the SE (n = 3). Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.01).
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4. Figure 2 BBX18-Mediated Thermomorphogenesis Requires PIF4
(A and B) PIF4 is epistatic to BBX18 and BBX23 in thermoregulated hypocotyl elongation. The bbx18-2 bbx23-2 pif4-101 triple mutant was generated by crossing the bbx18-2 bbx23-2 double mutant with the pif4-101 mutant.
(C–E) Dependence of PIF4 for the long-hypocotyl phenotype of BBX18-overexpression plants. BBX18-FLAG with the CaMV 35S promoter was overexpressed in either the wild-type (WT) or PIF4 mutant (pif4-101) background. Exogenous BBX18 protein accumulation was checked by western blotting with the anti-FLAG antibody. Tubulin served as a protein-loading control. The signal intensity of each band was quantified and normalized to that of the second sample (C). The results of hypocotyl length from two independent transgenic lines (BBX18ox) are shown in (D) and (E).
(F) PIF4 regulates thermomorphogenesis downstream of BBX18 and BBX23. PIF4 was overexpressed in the wild-type (WT) and bbx18-2 bbx23-2 double-mutant plants.
Four-day-old seedlings of transgenic lines in each genetic background grown at 22°C were transferred to either 22°C or 29°C conditions for 4 days, after which representative plants were imaged (A and D) and the hypocotyl lengths of each plant were measured (B, E, and F). Three biological replicates were performed, and eight plants were measured in each replicate. The bars depict the SE (n = 3). Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.01).
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5. Figure 3
BBX18, BBX23, and PIF4 Share Overlapping Downstream Genes during Thermomorphogenesis
(A) Upregulated or downregulated genes in the wild-type (WT), BBX18 and BBX23 double-mutant (bbx18-2 bbx23-2), and PIF4 mutant (pif4-101) plants in response to elevated ambient temperature. Six-day-old seedlings were transferred to 29°C, while the control seedlings were maintained at 22°C temperature. After 24 hr, the seedlings were collected for RNA-seq analysis from three biological replicates.
(B) Gene Ontology (GO) analysis of upregulated thermomorphogenesis-related genes that depend on PIF4, BBX18, and BBX23. The wild-type (WT), BBX18 and BBX23 double-mutant (bbx18-2 bbx23-2), and PIF4 mutant (pif4-101) plants were grown at 22°C or stressed for 24 hr at 29°C, after which they were sampled for RNA-seq.
(C) Validation of gene expression patterns obtained from RNA-seq by qRT-PCR. The relative gene expression levels were normalized to those of plants grown at 22°C, both of which were normalized to the expression levels of PP2A.
The bars depict the SE (n = 3). Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.01).
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6. Figure 4
Both BBX18 and BBX23 Interact with ELF3 and Regulate ELF3 Protein Accumulation at Elevated Ambient Temperature
(A–C) Protein-protein interactions between BBX18 and ELF3 or between BBX23 and ELF3. Yeast two-hybrid assays (A), in vitro pull-down assays (B), and in vivo split-luciferase assays (C) were performed.
(D and E) Epistatic effect of ELF3 on BBX18 and BBX23 for the thermoregulated hypocotyl elongation. The bbx18-2 bbx23-2 elf3-101 triple mutant was generated by crossing the bbx18-2 bbx23-2 double mutant with the elf3-101 mutant.
(F and G) Suppression of the hypocotyl phenotype of ELF3-overexpression plants in response to elevated ambient temperature by BBX18. BBX18-FLAG with the constitutive CaMV 35S promoter was overexpressed in Arabidopsis wild-type (WT) plants, and one of the lines (BBX18ox-10) was crossed with the ELF3-overexpression plants (ELF3ox-1).
(D and F) Four-day-old seedlings grown at 22°C were transferred to either 22°C or 29°C for 4 days, after which representative plants were imaged.
(E and G) The hypocotyl length of each plant was subsequently measured.
The PIF4 mutant (pif4-101) served as a control. Three biological replicates were performed, and eight plants were measured in each replicate. The bars depict the SE (n = 3). Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.01).
(H) ELF3 expression in the WT and BBX18/BBX23 double-mutant plants. Seven-day-old plant seedlings grown at 22°C were transferred to either 22°C or 29°C and then sampled at different time points (ZT0–ZT32) for total mRNA extraction and qRT-PCR. The relative gene expression is the expression of ELF3 in different samples normalized to that of the ZT8 sample at 22°C in the WT, all of which were normalized to the expression of the PP2A gene. The plants were grown under a 16/8-hour photoperiod (black and white bar; dark and light, respectively). The bars depict the SE (n = 3).
(I and J) Higher ELF3 protein accumulation in BBX18 and BBX23 double mutant than in WT at an elevated temperature.
Seven-day-old plant seedlings grown at 22°C were transferred to either 22°C or 29°C, after which they were sampled at ZT24 for western blotting. Histone H3 served as a protein-loading control. The signal intensity of each band was quantified and normalized to that of the first sample. The relative protein level is the quantified expression signals of ELF3 normalized to that of H3 from five western blots. Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.05).
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7. Figure 5 BBX18- and BBX23-Mediated Thermomorphogenesis Requires COP1
(A and B) Protein-protein interactions between BBX18 and COP1 or between BBX23 and COP1 in pull-down assays (A) and split-luciferase assays in tobacco leaves (B).
(C and D) Dependence on COP1 for the long-hypocotyl phenotype of BBX18-overexpression plants. BBX18-FLAG was overexpressed in the wild-type (WT) background, and one of the lines (BBX18ox-3) was crossed with the COP1 mutant (cop1-4). BBX18-FLAG-overexpression plants in the homozygous COP1 mutant background were obtained from the F2 segregating population. Four-day-old seedlings grown at 22°C were transferred to either 22°C or 29°C for 4 days, after which representative plants were imaged (C). The hypocotyl length of each plant was subsequently measured (D). Three biological replicates were performed, and eight plants were measured in each replicate. The bars depict the SE (n = 3). Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.01).
(E) Validation of BBX18-FLAG overexpression in plants by western blotting with anti-FLAG antibodies. Ponceau-stained Rubisco small subunit (RbCS) served as a protein-loading control. The signal intensity of each band was quantified and normalized to that of the second sample. Note that the total proteins are not equal between the two temperature conditions.
(F and G) ELF3 degradation regulated by BBX18 or BBX23 or COP1. ELF3-LUC was constitutively expressed in tobacco leaves with either the empty vector control, or COP1, or BBX18, or BBX23, or together with BBX18, BBX23, and COP1. A proteasome inhibitor, MG132, or the solvent control DMSO, was infiltrated into tobacco leaves 12 hr prior to imaging. The firefly luciferase (LUC) activity was visualized with a CCD camera (F). Six individual leaves for each of the combinations were imaged and quantified (G). The bars depict the SE (n = 6). Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.01).
(H and I) Comparable ELF3 protein accumulation in WT and COP1 mutant plants at an elevated temperature. Samples were prepared as in Figure 4H. Histone H3 served as a protein-loading control. The signal intensity of each band was quantified and normalized to that of the first sample. The relative protein level is the quantified expression signals of ELF3 normalized to that of H3 from five western blots. Letters above the bars indicate significant differences as determined by Duncan’s multiple range test (p < 0.05).
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